MyoD belongs to a family of transcription factors - the myogenic bHLH- which is required for the formation of skeletal muscle in the animal. The transcriptional activity of MyoD is regulated at the DNA-binding level, by post-translational modifications, and by interaction with several regulatory proteins. Association of MyoD with histone modifiers and chromatin remodeling complexes regulates its transcriptional activity. Therefore, the identification of the macromolecular complexes associated with MyoD becomes relevant to the understanding of the regulatory mechanisms underlying gene expression in skeletal muscle. In the past years, we have identified and functionally characterized proteins and noncoding RNAs that influence the ability of MyoD to promote transcription. ? The acetyltransferases p300 and PCAF are associated with MyoD and promote acetylation of the histones located at the chromatin regions bound by MyoD. Histone acetylation promotes gene transcription. Additionaly,PCAF acetylates MyoD itself and, in doing so, increases its ability to interact with specific-chromatin regions and favors recruitment and retention of the RNA polymerase II (PolII). The engagement of PolII on MyoD transcribed DNA is achieved by an independent,but functionally coherent, mechanism. We have isolated p68 and p72 RNA-binding proteins as MyoD- associated factors. Their depletion -obtained by RNA interference- in mouse myoblasts impairs proper muscle gene expression and the differentiation program. Interestingly, not all genes regulated by MyoD require p68/p72. p68 is recruited at transcriptional regulatory regions of a subset of muscle genes in differentiated skeletal muscle cells but is absent at the same sites in undifferentiated myoblasts. Chromatin immunoprecipitation experiments performed with cells depleted of p68/p72 have uncovered a role for p68 in directing the assembly of the transcriptome and chromatin remodeling factors. Decreasing the levels of p68/p72 results in reduced recruitment of the TATA-binding protein (TBP), PolII and the chromatin remodeling protein Brg-1 at muscle regulatory regions. As a consequence, nucleosome remodeling is disrupted, adn transcriptional initiation complexes do not assemble at these promoter regions. In the same MyoD macromolecular complex containing p68/p72, we have identified SRA, a noncoding RNA. Our results indicate that SRA is required to promote expression of the same genes regulated by p68/p72. By isolating novel components and clarifying the mechanistic role of those already identified, we will continue to define and investigate the functional relevance of macromolecular complexes that regulate histone modifications and chromatin conformation and transcription in skeletal muscle cells.
